Amino acids are of considerable economic interest since amino acids have many uses: thus, for example, L-lysine and L-threonine, L-methionine and L-tryptophan are necessary as fodder additives, L-glutamate as an additive to suppress L-isoleucine and L-tyrosine in the pharmaceutical industry, L-arginine and L-isoleucine as medicaments or L-glutamate, L-aspartate and L-phenylalanine as starting substances for the synthesis of fine chemicals.
A preferred method of producing these different amino acids is the biotechnical production by means of microorganisms such that in this manner the biologically-effective and optically-active forms of the respective amino acids are obtained and simple and inexpensive raw materials can be used. As microorganisms, for example, Corynebacterium glutamicum and its derivatives sap. Flavum and ssp. Lactofermentum (Liebl et al., Int J System Bacteriol 1991, 41: 255 to 260) in addition to Escherichia coli and related bacteria are used. These bacteria normally produce the amino acids but only in amounts required for growth so that no surplus amino acids are formed and can be recovered. This is because in the cells the biosynthesis of amino acids is controlled in many ways. As a consequence, there are already known various processes to increase the product formation by cutting out the control mechanisms. In these processes, for example, amino acid analogs are introduced to switch off the effective regulation of the biosynthesis. For example, a process has been used which is resistant to L-tyrosine analogs and L-phenylalanine analogs (JP 19037/1976 and 39517/1978). The processes also have been described in which bacteria resistant to L-lysine analogs or L-phenylalanine analogs have been used to suppress the control mechanisms (EP 0 205 849, GB 2 152 509).
Furthermore, microorganisms which have been constructed also by recombinant DNA-techniques obviate regulation of biosynthesis in that the gene which is coded in the no-longer feedback-inhibited key enzyme is cloned and expressed. For example, the recombinant L-lysine-producing bacterium with plasmid-coded feedback-resistant aspartate kinase is known (EP 0 381 527). In addition, a recombinant L-phenylalanine-producing bacterium with feedback-resistant prephenate dehydrogenase is described (JP 123475/1986, EP 0 488 424).
In addition, by overexpression of genes which do not code for feedback-sensitive enzymes in amino acid synthesis, increased amino acid yields are obtainable. Thus, for example, lysine formation can be improved by increased synthesis of the dihydrodipicolinate synthesis (EP 0 197 335). Increasingly, by increased synthesis of the threoninedehydratease, improved isoleucine formation is achieved (EP 0 436 886).
Further investigations in increasing amino acid production have been targeted on the improved availability of the cellular primary metabolites of central metabolism. Thus it is known that, by recombinant techniques, over-expression of the transketolase can bring about an improved product formation of L-tryptophan or L-tyrosine or L-phenylalanine (EP 0 600 463). Furthermore, a reduction of the phosphoenolpyruvate-carboxylase activity in Corynebacterium leads to improved formation of aromatic amino acids (EP 0 3331 145) whereas by contrast an increase in the phosphoenolpyruvate-carboxylase activity in Corynebacterium leads to increased recovery of amino acids of the aspartate family (EP 0 358 940).
During the growth and especially under amino acid production conditions, the tricarboxylic acid cycle must continuously and effectively be supplemented with C4 compounds, for example, oxalic acetate to replace intermediate products withdrawn for the amino acid biosynthesis. Until recently it has been thought that phosphoenolpyruvate-carboxylase was responsible for these so-called anaplerotic functions in Corynebacterium (Kinoshita, Biology of Industrial Micro-organisms 1985: 115 to 142, Benjamin/Cummings Publishing Company, London; Liebl, The Prokaryotes II, 1991 to 1171, Springer Verlag N.Y.; Vallino and Stephanopoulos, Biotechnol Bioeng 1993, 41: 633 to 646).
It has, however, now been found that phosphoenolpyruvate-carboxylase-negative mutants grow equally by comparison to the respective starting strains on all media (Peters-Wendisch et al., FEMS Microbiology Letters 1993, 112: 269 to 274; Gubler et al., Appl Microbiol Biotechnol 1994, 40: 857 to 863). These results indicate that the phosphoenolpyruvate-carboxylase is not essential for the growth and plays no role or only a small role for the anaplerotic reactions. Furthermore the aforementioned results indicate that in Corynebacterium another enzyme must be provided which is responsible for the synthesis of oxalacetate which is required for growth. Recently, indeed, a pyruvate-carboxylase activity has been found in permeablized cells of Corynebacterium glutamicum (Peters-Wendisch et al., Microbiology 1997, 143: 1095 to 1103). This enzyme is effectively inhibited by AMP, ADP and acetyl coenzyme A and in the presence of lactate as a carbon source is formed in increased quantities. Since one must conclude that this enzyme is answerable primarily for the satisfaction of the tricarboxylic acid cycle of growth, it was to be expected that an increase in the gene expression or the enzymatic activity would either give rise to no increase in the amino acids belonging to the aspartate or yield only an increase therein. Furthermore, it was to be expected that an increase in the gene expression or the enzymatic activity of the pyruvate-carboxylase would also have no influence on the production of amino acids of other families.